1. Technical Field
The present invention relates to the use of yeast strains to modify substrates via biooxidation. More particularly, the present invention relates to processes for converting certain substrates into alcohols or carboxylic acids utilizing yeast.
2. Background of Related Art
Aliphatic dioic acids, alcohols and compounds having combinations of alcohols and acids are versatile chemical intermediates useful as raw materials for the preparation of adhesives, fragrances, polyamides, polyesters, and antimicrobials. While chemical routes for the synthesis of long-chain α,ω-dicarboxylic acids are available, the synthesis is complicated and results in mixtures containing dicarboxylic acids of shorter chain lengths. As a result, extensive purification steps are necessary. While it is known that long-chain dioic acids can also be produced by microbial transformation of alkanes, fatty acids or esters, chemical synthesis has remained the preferred route, presumably due to limitations with the previously available biological approaches.
Several strains of yeast are known to excrete α,ω-dicarboxylic acids as a byproduct when cultured on alkanes or fatty acids. In particular, yeast belonging to the genus Candida, such as C. albicans, C. cloacae, C. guillermondii, C. intermedia, C. lipolytica, C. maltosa, C. parapsilosis, and C. zeylenoides are known to produce such dicarboxylic acids. (Agr. Biol. Chem. 35, 2033-2042 (1971).) In addition, various strains of the yeast C. tropicalis are known to produce dicarboxylic acids ranging in chain lengths from C11 through C18 as a byproduct when cultured on alkanes or fatty acids as the carbon source (Okino et al., B M Lawrence, B D Mookherjee and B J Willis (eds.), in Flavors and Fragrances: A World Perspective. Proceedings of the 10th International Conference of Essential Oils, Flavors and Fragrances, Elsevier Science Publishers BV Amsterdam (1988)), and are the basis of several patents as reviewed by Bühler and Schindler, in Aliphatic Hydrocarbons in Biotechnology, H. J. Rehm and G. Reed (eds), Vol. 169, Verlag Chemie, Weinheim (1984).
Studies of the biochemical processes by which yeasts metabolize alkanes and fatty acids have revealed three types of oxidation reactions: α-oxidation of alkanes to alcohols; ω-oxidation of fatty acids to α,ω-dicarboxylic acids; and the degradative β-oxidation of fatty acids to CO2 and water. In C. tropicalis the first step in the ω-oxidation pathway is catalyzed by a membrane-bound enzyme complex (ω-hydroxylase complex) including a cytochrome P450 monooxygenase and a NADPH-cytochrome reductase. This hydroxylase complex is responsible for the primary oxidation of the terminal methyl group in alkanes and fatty acids (Gilewicz et al., Can. J. Microbiol. 25:201 (1979)). The genes which encode the cytochrome P450 and NADPH reductase components of the complex have previously been identified as P450ALK and P450RED respectively, and have also been cloned and sequenced (Sanglard et al., Gene 76:121-136 (1989)). P450ALK has also been designated P450ALK1. More recently, ALK genes have been designated by the symbol CYP and RED genes have been designated by the symbol CPR. See, e.g., Nelson, Pharmacogenetics 6(1):1-42 (1996), which is incorporated herein by reference. See also Ohkuma et al., DNA and Cell Biology 14:163-173 (1995), Seghezzi et al., DNA and Cell Biology, 11:767-780 (1992) and Kargel et al., Yeast 12:333-348 (1996), each incorporated herein by reference. For example, P450ALK is also designated CYP52 according to the nomenclature of Nelson, supra.
Cytochromes P450 (P450s) are terminal monooxidases of the multicomponent enzyme system described above. They comprise a superfamily of proteins which exist widely in nature having been isolated from a variety of organisms, e.g., various mammals, fish, invertebrates, plants, mollusks, crustaceans, lower eukaryotes and bacteria (Nelson, supra). First discovered in rodent liver microsomes as a carbon-monoxide binding pigment as described, e.g., in Garfinkel, Arch. Biochem. Biophys. 77:493-509 (1958), which is incorporated herein by reference, P450s were later named based on their absorption at 450 nm in a reduced-CO coupled difference spectrum as described, e.g., in Omura et al., J. Biol. Chem. 239:2370-2378 (1964), which is incorporated herein by reference.
P450s catalyze the metabolism of a variety of endogenous and exogenous compounds (Nelson, supra). Endogenous compounds include steroids, prostanoids, eicosanoids, fat-soluble vitamins, fatty acids, mammalian alkaloids, leukotrines, biogenic amines and phytolexins (Nelson, supra). P450 metabolism involves such reactions as aliphatic hydroxylation, aromatic oxidation, alkene epoxidation, nitrogen dealkylation, oxidative deamination, oxygen dealkylation, nitrogen oxidation, oxidative desulfuration, oxidative dehalogenation, oxidative denitrification, nitro reduction, azo reduction, tertiary amine N-oxide reduction, arene oxide reduction and reductive dehalogenation. (P G Wislocki, G T Miwa and AYH Lu, Reaction Catalyzed by the Cytochrome P-450 System, Enzymatic Basis of Detoxication, Vol. 1, Academic Press (1980).) These reactions generally make the compound more water soluble, which is conducive for excretion, and more electrophilic. (These electrophilic products have detrimental effects if they react with DNA or other cellular constituents.) The electrophilic products can then react through conjugation with low molecular weight hydrophilic substances resulting in glucoronidation, sulfation, acetylation, amino acid conjugation or glutathione conjugation typically leading to inactivation and elimination as described, e.g., in Klaassen et al., Toxicology, 3rd ed, Macmillan, New York, 1986, incorporated herein by reference.
Fatty acids are ultimately formed from alkanes after two additional oxidation steps, catalyzed by alcohol oxidase (Kemp et al. Appl. Microbiol. and Biotechnol, 28, 370-374 (1988)) and aldehyde dehydrogenase. The, ω-hydroxylase enzymes of the ω-oxidation pathway are located in the endoplasmic reticulum, while the enzymes catalyzing the last two steps, the fatty alcohol oxidase and the fatty aldehyde dehydrogenase, are located in the peroxisomes. The fatty acids can be further oxidized through the same or similar pathway to the corresponding dicarboxylic acid. The ω-oxidation of fatty acids proceeds via the ω-hydroxy fatty acid and its aldehyde derivative, to the corresponding dicarboxylic acid without the requirement for CoA activation. However, both fatty acids and dicarboxylic acids can be degraded, after activation to the corresponding acyl-CoA ester through the β-oxidation pathway in the peroxisomes, leading to chain shortening. In mammalian systems, both fatty acid and dicarboxylic acid products of ω-oxidation are activated to their CoA-esters at equal rates and are substrates for both mitochondrial and peroxisomal β-oxidation (J. Biochem., 102, 225-234 (1987)). In yeast, β-oxidation takes place solely in the peroxisomes (Agr. Biol. Chem., 49, 1821-1828 (1985)).
Metabolic pathways can be manipulated in an attempt to increase or decrease the production of various products or by-products. Knowing that fatty acids possessing one or more internal double bonds or secondary alcohol functionality are capable of undergoing ω-oxidation, the ω-oxidation pathway can be manipulated to produce greater amounts of dicarboxylic acids. U.S. Pat. No. 5,254,466, the entire contents of which are incorporated herein by reference, discloses a method for producing β,ω-dicarboxylic acids in high yields by culturing C. tropicalis strains having disrupted chromosomal POX4A, POX4B and both POX5 genes. The POX4 and POX5 gene disruptions effectively block the β-oxidation pathway at its first reaction (which is catalyzed by acyl-CoA oxidase) in a C. tropicalis host strain. The POX4 and POX5 genes encode distinct subunits of long chain acyl-CoA oxidase, which are the peroxisomal polypeptides (PXPs) designated PXP-4 and PXP-5, respectively. The disruption of these genes results in a complete block of the β-oxidation pathway thus allowing enhanced yields of dicarboxylic acid by redirecting the substrate toward the ω-oxidation pathway and also preventing reutilization of the dicarboxylic acid products through the β-oxidation pathway.
Similarly, C. tropicalis may also have one or more cytochrome P450 genes and/or reductase genes amplified which results in an increase in the amount of rate-limiting ω-hydroxylase through P450 gene amplification and an increase in the rate of substrate flow through the ω-oxidation pathway. C. tropicalis strain AR40 is an amplified H 5343 strain wherein all four POX4 genes and both copies of the chromosomal POX5 genes are disrupted by a URA3 selectable marker and which also contains 3 additional copies of the cytochrome P450 gene and 2 additional copies of the reductase gene, the P450RED gene. Strain AR40 has the ATCC accession number ATCC 20987. C. tropicalis strain R24 is an amplified H 5343 strain in which all four POX4 genes and both copies of the chromosomal POX5 genes are disrupted by a URA3 selectable marker and which also contains multiple copies of the reductase gene. Strains AR40 and R24 are described in U.S. Pat. Nos. 5,620,878 and 5,648,247, the contents of which are incorporated herein by reference.
Processes for utilizing modified C. tropicalis to produce carboxylic acids are also known. U.S. Pat. No. 5,962,285, the entire contents of which are incorporated herein by reference, discloses a process for making carboxylic acids by fermenting a β-oxidation blocked C. tropicalis cell in a culture comprised of a nitrogen source, an organic substrate and a cosubstrate. The substrate is an unsaturated aliphatic compound having at least one internal carbon-carbon double bond and at least one terminal methyl group, a terminal carboxyl group and/or a terminal functional group which is oxidizable to a carboxyl group. The fermentation product is then reacted with an oxidizing agent to produce one or more carboxylic acids.
Similar shake flask experiments have been used in the past to test substrates. The terminal methyl group and the terminal double bond of α-alkenes or branched monoacids are oxidized and form alcohol groups or the desired acid groups. The oxidation of the terminal double bond of α-olefins to form a (ω,ω-1) diol is an interesting reaction. The overall oxidation product is thus a (ω,ω-1) hydroxyfatty acid. The biooxidation of α-olefins was first reported by Uemura. (N. Uemura, Industrialization of the Production of Dibasic Acid from n-Paraffins Using Microorganisms, Hakko to Kogyo, 43:436-44 (1985).).
While the genetically modified strains of Candida sp. are able to produce large quantities of product necessary to develop a commercially feasible process, it is not known what effect variations of chain length, functional groups, etc. will have on the ability of C. tropicalis to produce alcohols and carboxylic acids through the process of biooxidation.